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Sonication mediated covalent cross-linking ofDNA to single-walled carbon nanotubesBridget D. DolashBirck Nanotechnology Center, Purdue University

Roya R. LahijiBirck Nanotechnology Center, Purdue University

Dmitry Y. ZemlyanovBirck Nanotechnology Center, Purdue University, dimazemlyanov@purdue.edu

Vladimir P. DrachevBirck Nanotechnology Center, Purdue University, vdrachev@purdue.edu

Ronald ReifenbergerBirck Nanotechnology Center, Purdue University, reifenbr@purdue.edu

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Dolash, Bridget D.; Lahiji, Roya R.; Zemlyanov, Dmitry Y.; Drachev, Vladimir P.; Reifenberger, Ronald; and Bergstrom, Donald E.,"Sonication mediated covalent cross-linking of DNA to single-walled carbon nanotubes" (2013). Birck and NCN Publications. Paper1345.http://dx.doi.org/10.1016/j.chemphys.2012.07.004

AuthorsBridget D. Dolash, Roya R. Lahiji, Dmitry Y. Zemlyanov, Vladimir P. Drachev, Ronald Reifenberger, andDonald E. Bergstrom

This article is available at Purdue e-Pubs: http://docs.lib.purdue.edu/nanopub/1345

Sonication mediated covalent cross-linking of DNA to single-walledcarbon nanotubes

Bridget D. Dolash a,b, Roya R. Lahiji b,c, Dmitry Y. Zemlyanov b, Vladimir P. Drachev b,d,⇑,Ronald Reifenberger b,c, Donald E. Bergstrom a,b

a Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USAb Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907, USAc Department of Physics, Purdue University, West Lafayette, IN 47907, USAd School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907, USA

a r t i c l e i n f o

Article history:Available online 20 July 2012

Keywords:Carbon nanotubesSonicationDNACovalent cross-linkingFree radicals

a b s t r a c t

Sonication with nucleic acids has become a standard method for obtaining aqueous dispersions of carbonnanotubes. On the basis of theoretical studies and scanning probe microscopy (SPM) imaging a widelyaccepted model for DNA association with SWCNT is one in which the DNA binds through non-covalentp-stacking and hydrophobic interactions. Following the standard procedures established by others toprepare DNA associated single-wall carbon nanotubes (SWCNT), we have determined that sonicationgenerates radical intermediates then form covalent anchors between the DNA and SWCNT. In light of thisfinding, results from studies on DNA associated carbon nanotubes, need to be more carefully interpreted.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

DNA-associated single-walled carbon nanotubes (SWCNTs)have been studied extensively since Zheng et al. reported thehybrids in 2003 [1,2]. These conjugates form an important bridgebetween nanotechnology and biology. Both experimental dataand theoretical studies have provided a solid foundation for under-standing the nature of these interactions [2–5]. Theoretical studiessuggest the primary forces responsible for the hybrids are throughp-stacking and hydrophobic interactions [6,7]. The influence ofboth DNA sequence and CNT structure (e.g. size and chirality) havebecome topics of intense investigation [8,9].

Despite extensive investigation the nature of the interactionsarising involving DNA during the conjugation process have notbeen fully resolved. The common method for the conjugation in-volves sonication of the SWCNTs in the presence of DNA in aque-ous solutions [1,2]. Sonication has been known to producehydroxyl (OH�) and peroxide (HOO�) radicals in aqueous solutions[10,11]. These radicals then produce base modifications, primarilyby radical oxidation, on DNA as well as single strand breaks [12–15]. As a result, DNA associated with SWCNTs may be damaged.In addition, it is known that free radicals react with SWCNTs allow-

ing cross-linking to various molecules [16]. The question thenarises as to whether DNA may become covalently cross-linked toSWCNTs following non-covalent association and sonication.

There are a considerable number of studies on the covalentmodification of SWCNT sidewalls by radical reactions. The reac-tions include addition of phenyl radicals generated from diazo-nium salts [17] or decarboxylation of benzoyl peroxide [18] andradicals generated by the sonochemical decomposition of o-dichlo-robenzene [19]. It is also clear from studies on both SWCNT andMWCNT that they are avid scavengers of hydroxyl radicals gener-ated by Fenton oxidation of H2O2 [20], sonication [21], or high volt-age-triggered arc discharges in aqueous suspension of CNTs [22].More recently both experimental and theoretical studies haveestablished that the reaction of hydroxyl radical with CNT is highlydependent on CNT structure with metallic carbon nanotubes morereactive than semiconducting nanotubes, and smaller diametertubes more reactive than larger diameter [23,24]. Perhaps the mostwell characterized reaction leading to covalent cross-links is thereaction of phenyl diazonium reagents with carbon nanotubes.Strano and coworkers have established that metallic carbon nano-tubes are more reactive than semiconducting [25,26]. A significantnumber of reports have established the reversibility of covalentcross-linking using phenyl radical conjugation as a model[27,28]. Just as different types (metallic, semiconducting, chirality,size) of carbon nanotubes show differences in reactivity, the kinet-ics of the reverse process, which regenerates pristine CNT, dependson the structure of the CNT to which a phenyl group is covalently

0301-0104/$ - see front matter � 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.chemphys.2012.07.004

⇑ Corresponding author at: Birck Nanotechnology Center, Purdue University,West Lafayette, IN 47907, USA.

E-mail addresses: vdrachev@purdue.edu (V.P. Drachev), bergstrom@purdue.edu(D.E. Bergstrom).

Chemical Physics 413 (2013) 11–19

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linked. The picture is further complicated because of differences inreactivity at different types of defect sites [24], which are com-monly found in single-wall carbon nanotubes.

It is well established that sonication of water generates the rad-icals HO and H, which oxidize organic materials [29]. The closestanalogy to CNT sonochemical degradation may be the sonochemi-cal mediated oxidation of polycyclic aromatic hydrocarbons, thepathway of which is still not well established [30], However, ithas been proposed that the major path for degradation may be ahigh temperature pyrolysis reaction that occurs within cavitationbubbles yielding radical cation intermediates [31]. The reactionoutcome (incorporation of hydroxyl groups through reaction ofwater with the cation) would be similar to that facilitated by directattack of hydroxyl radicals. Interestingly, free radicals scavengersinhibit the oxidation. The sonochemical degradation of CNT canbe imagined to follow the same pathway and indeed, studies onsonication of CNT show appearance of C–OH, then C@O, followedby CO2H by infrared spectroscopy [21].

A previous study from our labs used scanning probe microscopyto characterize SWCNTs associated with polythymidine DNA (T30)[32]. The study verified that sonication of SWCNTs in the presenceof T30 in aqueous buffer creates a deep grey/black solution. The solu-tion concentrations as well as the degree of T30 association werecharacterized using ultraviolet–visible (UV–Vis) spectroscopy.Scanning probe microscopy (SPM) was used to characterize theT30:SWCNT conjugates and verified that there is association be-tween the molecules. In this study, we report a series of experimentsto establish DNA can become covalently attached to the SWCNTs.

2. Results

With interest in biological applications of DNA-SWCNTs, we ex-plored the use of fluorescein modified T30 DNA (30 2’-deoxythymi-dine residues linked by 3’–5’ phosphodiester groups) conjugated toSWCNTs (Flu-T30: SWCNT) for fluorescent imaging purposes.However, in comparison to T30 alone, Flu-T30 failed to solubilizeSWCNTs as seen visually by a lack of grey/black color in solution.The absence of Flu-T30: SWCNTs was confirmed by fluorescencespectroscopy and by very low absorbance from 190 to 800 nm asread by UV–Vis spectroscopy (data not shown). In contrast, T30without attached fluorescein, gave a deep grey/black dispersionthat, following centrifugation, remained stable over a long periodof time. The generation of free radicals via sonication (as discussedabove) taken together with reports of fluorescein acting as aquencher of free radical reactions [33], suggested that perhaps itwas the free radical inhibiting properties of the fluorescein thatinhibited the solubilization of the SWCNTs by the T30. An alterna-tive explanation that the fluorescein was simply interfering withthe wrapping of the T30 around the SWCNT seemed less plausiblegiven that only a single fluorescein molecule was attached at oneend of the T30. Furthermore there are no reports in the literatureof other fluorescent molecules interfering with DNA solubilization.This led to the hypothesis that DNA may be causing radical reac-tions leading to a covalent anchor between the two molecules. Inthe presence of fluorescein the free radical reaction is inhibited,cross-linking does not occur, and non-covalent association is insuf-ficient to facilitate DNA: SWCNT dispersion. Consequently, we de-signed a set of experiments to test this hypothesis.

2.1. Effect of free radical inhibitors on the sonication-mediateddispersion of SWCNTs by DNA

To further investigate this hypothesis of cross-linking, the effectsof two common free radical scavengers on DNA-SWCNT formationwere studied. Namely, (±)-6-hydroxy-2,5,7,8-tetramethylchro-

mane-2-carboxylic acid (Trolox, a vitamin E derivative) and ascorbicacid (vitamin C) were selected. Each was added in the initial reactionmixture, which consisted of SWCNTs and DNA (non-fluorescein con-jugated) in PIPES buffer solution. The final solutions were character-ized by UV–Visible spectroscopy by obtaining the absorbance valueat 750 nm and using an extinction coefficient of 488 mol C L�1 cm�1

[34]. As shown in Table 1, T30 disperses significantly more SWCNTsthan A30 (an oligonucleotide consisting of 30 adenosine nucleo-tides), which is supportive of Zheng et al. [1]. It is also clear thatthe addition of either Trolox or ascorbic acid significantly reducesthe amount of dispersed SWCNTs. Ascorbic acid reduced the disper-sion to <1% of the amount dispersed in the absence of free radicalscavengers, which appears to be more effective than Trolox, whichreduced the dispersion between 4–7%. Similar results were also seenwith an alternating thymine–guanine 30-mer sequence (TG15) aswell as a self-complementary DNA sequence (50-TGT GTG TGT GTGTGT ACA CAC ACA CAC ACA-30) used for dispersion of SWCNTs as thisassociation was also strongly inhibited by ascorbic acid (data notshown). Dispersion of SWCNTs by T30 and TG15 were also reducedby Trolox in alternative buffers including 0.1 M NaCl, 1 M NaCl andphosphate buffered saline (PBS).

It is important to verify that the inhibition of DNA dispersion byTrolox is due to inhibition of free radical reactions as opposed tocompetition of Trolox molecules with DNA for non-covalent asso-ciation with the SWCNT. To demonstrate this property, an experi-ment was carried out in which the Trolox was replaced by (S)-Trolox methyl ether. Unlike Trolox in which the phenolic hydroxylis the source of a hydrogen atom for quenching radical reactions,(S)-Trolox methyl ether has no phenolic hydroxyl group. As a re-sult, it is not a free radical inhibitor but it retains the high lipophil-icity of Trolox and would be expected to have a similar affinity fornon-covalent association. When T30 and SWCNTs were sonicatedin the presence of (S)-Trolox methyl ether, the amount of SWCNTsin solution was comparable to T30:SWCNTs conjugated in the ab-sence of Trolox. This result is supportive that prevention ofDNA:SWCNT dispersion by Trolox is due to free radical inhibition.

If the DNA is covalently bound to the SWCNTs, it seemed possi-ble that it could retain its ability to hybridize to a complementarysequence. Indeed, based on our SPM studies [32], the majority ofthe SWCNT had ‘‘bare’’ regions that were free of nucleic acids. Con-sequently, the addition of Flu-T30 or Flu-A30 to such dispersedDNA:SWCNT solution should lead to association of either the Flu-T30 or Flu-A30 to the T30:SWCNT. If the T30 is covalently linkedto the SWCNT and non-covalent association is weak, then onlyFlu-A30 should show significant association with the T30:SWCNT(due to complementary hybridization), Flu-T30 would not hybrid-ize and should be removed by washing.

A solution of Flu-A30 DNA was mixed with T30:SWCNT:120 for1 h, centrifuged and filtered. We observed no significant precipita-tion of carbon nanotubes in solution. Fluorescence spectroscopyshowed the presence of fluorescein. To verify that the hybridiza-tion is dependent on the presence of complementary DNA, acontrol was carried out using Flu-T30 DNA. The amount of fluores-cein as detected by fluorescence spectroscopy was dramaticallydecreased suggesting that the complementary sequence is

Table 1Effect of free radical inhibitors on DNA:SWCNT dispersion.

Sample Relative dispersiona

T30:SWCNT 100A30:SWCNT 51.4T30:SWCNT + Vitamin C 0.9A30:SWCNT + Trolox 7.3T30:SWCNT + Trolox 4.4

a Percentage of carbon mass dispersed in solution relative to T30:SWCNT.

12 B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19

necessary for the presence of fluorescein-conjugated DNA (Table 2).This data supports a model in which the T30 is covalently bound toSWCNT at sufficiently few sites (possibly only one) that the non-covalently bound region of the T30 is free to bind to the comple-mentary sequence (A30).

2.2. Characterization of chemical changes to SWCNTs duringsonication

In order to assess the change in chemical composition ofSWCNT caused by sonication, X-ray photoelectron spectroscopy

(XPS) was used. XPS is a surface sensitive technique that has be-come widely used for studying properties of atoms, molecules, sol-ids, and surfaces. Generally speaking, intensities of core levelphotoelectron peaks are used for quantitative analysis, and bindingenergies of core level photoelectrons typically exhibit chemically-induced shifts [35]. Since CNT has aromatic structure, a shake-upsatellite is one of the important characteristics of CNT. Theshake-up satellites are typical photoemission process in aromaticsystems, which is a two electron phenomenon leading to p ? p⁄

transition involving the highest filled and lowest unfilled valencelevels. Aromatic systems show shake-up peaks with intensities ofup to 5–10% [35]. Damage of nanotubes caused by oxidation [36]or by Ar+ bombardment [37] leads to a decrease/disappearance ofthe p–p⁄ peak. If sonication is resulting in the partial destructionof the extended p-system through reactions mediated by the highenergy associated with bubble collapse on sonication then thisband should decrease in intensity relative to the main C 1s peak.

Three samples were studied by XPS, as-received SWCNTs(ar-SWCNTs), SWCNTs after sonication in water (SWCNT:120),and SWCNTs after sonication in water in the presence of Trolox(SWCNT:120 + Trolox). As summarized in Table 3 the C:O ratio de-creases on sonication of the SWCNT in aqueous solution. The highpercentage of oxygen in the ar-SWCNT reflects the processing bythe manufacturer, in which metal contaminates are typically re-moved by nitric acid oxidation. As seen in Fig. 1, SWCNT:120s showa rise in the C–O (�287 eV) C 1s component. At the same time therelative intensity of the shake-up satellite is decreased indicatingdeterioration of the extended p-system. The deterioration in wall

Table 2Hybridization of DNA:SWCNTs to complementary DNA.

Sample Fluorescence intensity

T30:SWCNT + Fl-A30 154.74T30:SWCNT + Fl-T30 54.51

Table 3Effect of sonication on SWCNTS as probed by XPS.

Sample C:O ratio C 1s components

C–C% C–O% O@C–OH% Shake-up%

ar-SWCNTs 25.3 83 5 2 10SWCNT:120 11.0 85 7 2 7SWCNT:120 + Trolox 7.3 88 7 2 3

Fig. 1. X-ray photoelectron spectra (XPS) of sonicated SWCNTs. (a) Binding energy plot for as-received SWCNT in the region 282–294 eV, (b) binding energy plot for SWCNTsonicated in aqueous solution for 120 min, and (c) binding energy plot for SWCNTs sonicated in aqueous solution in the presence of Trolox.

B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19 13

structure can also be clearly seen in the high resolution TEM imagesof the SWCNT:120 [38,39]. Interestingly, the SWCNT:120 + Troloxshow a similar increase in the percentage of incorporated oxygen.Significantly it is the C–O component that is increased rather thanthe O–C@O component (Table 3). The p–p⁄ satellite is also consid-erably decreased.

The increase in the C–O component raised concern that theTrolox was facilitating an increase in oxidation leading to destruc-tion of the sidewalls of the CNTs. Extreme destruction of the side-wall could prevent p-stacking interactions and wrapping of theDNA about the SWCNT. In order to test this hypothesis, SWCNTswere sonicated in the presence of Trolox for 120 min. The nano-tubes were then filtered over a membrane filter to remove any freeTrolox. The filtered SWCNT:120 + Trolox were then sonicated inthe presence of T30 for 120 min (T30:SWCNT after Trolox). A blacksolution after purification indicated the CNT sidewalls were intactand available for association with T30.

Raman spectroscopy has emerged as one of the most commontechniques used for characterization of carbon nanotubes [40].The spectra contain common features for carbon materials includ-ing the G band and the disorder (D) band. The G band is indicativeof all pairs of sp2 atoms in both rings and chains. This band is splitinto two bands for nanotubes, the G band that represents displace-ment along the CNTs axes and the G� band that represents circum-ferential displacement. The D band arises due to the breathingmodes of sp2 atoms in rings making it an indicator of defects inthe CNTs as well as bundling effects. A feature in the Raman

spectra of nanotubes is the radial breathing mode (RBM) of thetube as a whole. The RBM is affected by diameter and chirality ofthe CNTs. We have employed Raman spectroscopy to identify thepresence of changes to the carbon nanotubes, if any, induced bysonication.

The half-height width of the asymmetric, Breit-Wigner-Fanoline (G� band) in the region 1450–1650 cm�1 is reduced in solutionrelative to the spectra for the samples dried onto filters on excita-tion at 514.5 nm (Fig. 2a). The half-height line widths are muchmore reduced on excitation at 647 nm (Fig. 2b). These results areconsistent with the results reported in the literature, where suchchanges were attributed to the bundling/debundling effect as wellas the localization or polarization of charges on the nanotube in-duced by molecular interactions [41,42]. Spectra normalized tothe G-band show increases in D-band (1280–1340 cm�1) intensityafter sonication as seen in Fig. 2a comparing ar-SWCNT (red line)with T30:SWCNT (purple line) and T30:SWCNTs after Trolox(green line). This result is suggestive of an increased number of de-fect sites as well as changes in the bundling effect. Interestingly,the D-band intensity increases as the amount of sonication in-creases, supporting the hypothesis of increased ‘‘disorder’’ or side-wall modification during the sonication process (Fig 2a and b).When the sample is observed in solution, the D-band intensity is

Fig. 2. Raman Spectra of treated SWCNTs. T30:SWCNTs (purple), T30:SWCNTs afterTrolox dried onto a filter (green), T30:SWCNTs after Trolox in solution (blue), andar-SWCNTs (red) were excited at 514.5 nm (a) and 647 nm (b) with 1.4 mW. Thespectra have been normalized to the G-band intensity and the background has beensubtracted. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

Fig. 3. Radial Breathing Mode (RBM) as obtained from Raman Spectroscopy.Samples were excited at 514.5 nm with 1.4 mW. The spectra have been normalizedto the G-band and the background has been subtracted. (a) RBM frequenciesincrease when nanotubes are treated as seen by T30:SWCNTs (purple) andT30:SWCNTs after Trolox (green) when compared to ar-SWCNTs. A more pro-nounced effect is seen when T30:SWCNTs after Trolox are observed in solution(blue) and (b) Sonication increases RBM frequencies relative to ar-SWCNTs (red)when sonicated in the presence (brown) and absence (blue) of Trolox. A similarincrease is seen with T30:SWCNTs (turquoise). (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of thisarticle.)

14 B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19

decreased dramatically when compared to a dry sample, suggest-ing that monitoring this parameter would be sensitive when insamples are in solution. This observation is supportive of biologicalapplications in aqueous media and the use of Raman spectroscopyto detect CNTs in biological samples [43]. It is also important tonote that it has been established that the ratio of the D–G bandintensities is indicative of covalent cross-linking of molecules toSWCNTs [26].

Second, RBM mode frequencies are higher for SWCNT:120than for ar-SWCNTs. It was similarly observed that SWCNTs insolution have higher RBM frequency modes than those driedonto a filter. Both bundling and surfactants can cause shifts inthe RBM frequencies. We were able to observe these effectsand attribute them to the presence of T30 acting as a surfac-tant. Eq. (1) has been a proposed form for the RBM frequency:[44].

xcalcRBM ¼ A=dt þ Bþ ðC þ D cos2 3hÞ=d2

t ð1Þ

The equation shows the RBM is dependent on the tube diameterdt and chirality angle h. The A constant describes the elasticbehavior of an isolated SWCNT with the theory predictedA = 227 cm�1 for both semiconductor and metallic SWCNT. TheB constant accounts for the interaction between the SWCNT ina bundle and between the SWCNT and surfactant. The valuesfor B have been reported as 7.3 cm�1 and 11.8 cm�1 for semicon-ducting and metallic nanotubes respectively. The C and D con-stants have negative values and account for chirality dependentcurvature effects. Their values are in the range of �1 to�3 cm�1. Our results show the Raman spectra of SWCNTs in solu-tion have higher frequencies of the RBM peaks (by 1.5 cm�1) eventhough the bundling effect does not contribute there (Fig. 3).Among the dry samples, the ar-SWCNTs should have a bundlingeffect but no effect by the DNA. As a result it has the frequenciesless than all other samples (Fig. 3).

3. Discussion

Based on, (a) the absence of dispersion of carbon nanotubes byT30 in the presence of free radical inhibitors, (b) the details of thestructure of T30 dispersed SWCNTs by SPM [32], (c) Raman andXPS data, and (d) the specific hybridization of T30 associatedSWCNTs with A30 without loss of dispersion, it appears plausiblethat sonication leads to the generation of covalent linkages be-tween oligonucleotides and SWCNTs. Although much more exten-sive studies will be required to establish both the sites of linkageand the mechanism for the cross-linking reactions it is worthwhileto set out working hypotheses here in order to guide the design offuture experiments. There are at least three general pathways bywhich sonication-mediated cross-linking can occur. These include:

(1) Generation of CNT radical cations by sonication-mediatedpyrolysis followed by reaction of the CNT radical cation with oligo-nucleotides (paths a and b, Fig. 4).

(2) Generation of reactive oxygen species (e.g. hydroxyl radi-cals) that react with CNTs to yield intermediate CNT radicals,which subsequently react with oligonucleotides (path c, Fig. 5).

(3) Reaction of reactive oxygen species with oligonucleotides togenerate oligonucleotide radicals or radical cations that react withCNTs.

While our experiments to establish cross-linking do not elimi-nate any of the hypothesized pathways, they do suggest prefer-ences. The first of the three hypothesized cross-linkingmechanisms, illustrated in Fig. 4 is initiated by pyrolysis of theSWCNT generating radical cations. From this intermediate, twopathways for the addition are illustrated. In path a, an electronfrom the CNT radical cation and an electron from the 5,6-doublebond of thymine combine to generate a carbon–carbon single bondbetween the CNT and the oligonucleotide. The CNT cation and

Fig. 4. Mechanism for sonication mediated covalent cross-linking of T30:SWCNTs. Sonication of SWCNTs in aqueous buffer creates radical cations on the nanotube sidewalls,which then react with water and T30. The addition to T30 may occur either through radical to the C-5,6 double bond (path a) or alkylation of a nucleophilic oxygen (path b).

B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19 15

water react with loss of a proton to introduce a hydroxyl group.Alternatively, as illustrated in path b, the CNT radical cation couldreact as an electrophile, alkylating T30 at any of a number of nucle-ophilic sites, including O2 or O4 of thymine [45], or phosphodiesteroxygen. The remaining CNT unpaired electron is likely to be unusu-ally stable because of extensive resonance through the CNT p-sys-tem, but ultimately will be annihilated through radicalrecombination or transfer reaction. As more CNT carbon oxygenbonds are generated, the complexity of the radical reactions willincrease, ultimately leading to CNT fragmentation concomitantwith oligonucleotide cross-linking.

The second alternative (path c, Fig. 5), involves sonication med-iated generation of reactive oxygen species (hydroxyl and hydro-peroxy radicals), followed by reaction of the reactive oxygenspecies with the CNT to generate the CNT radical. As illustratedin Fig. 5, a hydroxyl radical attacks a CNT followed by addition ofthe CNT radical to C5, 6 double bond of thymine. The mechanismby which the oligonucleotide reacts with the CNT radical is virtu-ally identical in paths a and c. The two paths differ only in theway in which a CNT radical is generated. The reaction would befacilitated by close non-covalent association of the T30 moleculewith the CNT. A third mechanistic pathway (not shown) would

be complex because of the variety of radical species that are gener-ated on reaction of DNA with reactive oxygen species.

Trolox and ascorbic acid are highly reactive towards radicalsand radical cations [46,47] and at high concentration will rapidlyquench reactive oxygen species (including hydroxyl radical) thatwould otherwise react with the SWCNT. It seems as likely thatTrolox and ascorbic acid could quench radicals and radical cationsthat form on carbon nanotubes. In the case of Trolox, quenchingcould occur by transfer of an electron to the CNT radical cation(path d, Fig. 6) to yield the Trolox radical cation, which would rap-idly lose a proton to water [48], to generate the stable Trolox rad-ical. Thereby CNT damage is mitigated. Alternatively, the CNTradical cation may react by path e in which a hydrogen atom fromTrolox and an OH from water become attached to the CNT. Troloxradical could also become covalently linked to the CNT by radicalrecombination (path f). As illustrated in Fig. 6, Trolox radicals canbe formed not only by paths d and e, but also by reaction of Troloxwith any radical species generated by sonication. Path g illustratesthe generation of Trolox radical from hydrogen and hydroxylradicals.

Addition of Trolox to the CNT sidewall is supported by increasedC–O content as seen in the XPS data as discussed above. If direct

Fig. 5. Alternative mechanism for sonication mediated covalent cross-linking of T30:SWCNTs. Sonication in aqueous buffer creates hydroxyl and hydrogen radicals, whichadd to carbon nanotube sidewalls to generate CNT radicals, which in turn add to associated, but non-covalently bound T30. This is a variation of path a in Fig. 6, differing onlyin the source of the CNT radical.

Fig. 6. Trolox reactions mediated by sonication in the presence of SWCNTs. Sonication generates CNT radical cations, which can react with Trolox by either path d or e. Bothpaths lead to stable Trolox radicals. Alternatively Trolox can intercept hydrogen (H�) and hydroxyl (OH�) radicals generated by sonication of H2O to generate Trolox radical(path f). Reaction of Trolox radical with CNT radical cation leads to a covalent link between the Trolox and SWCNT (path g).

16 B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19

radical addition were the predominant path (Fig. 5), then the rad-ical inhibitors should block the incorporation of oxygen into theSWCNT. However, if the generation of radical cations is the primarypathway, the radical quenching does not block the reaction of thecationic sites by nucleophiles, so the relative content of oxygenshould increase when the SWCNT are sonicated, both in the pres-ence and absence of radical inhibitors. This outcome can be ex-plained by the mechanism outlined in Fig. 6. The radical inhibitorwould not be expected to inhibit the formation of CNT radical cat-ions caused by high energy collisions mediated by bubble collapseon sonication. On the other hand, the radical inhibitor would be ex-pected to intercept the radical once it is formed and prevent car-bon–carbon bond fragmentation reactions that are a commonoutcome of radical cation species. In this case the oxygen incorpo-ration would be limited to nucleophilic attack by water or otheroxygen containing nucleophile generating C–O bonds. Formationof C@O bonds is limited by the lack of the radical mediated C–Cbond cleavage reactions that are required to generate them fromC–OH or C–O–C functional groups.

The existence of non-covalent interactions between DNA andSWCNTs is well-established. However, these interactions do notin any way preclude simultaneous covalent cross-linking facili-tated by radical species generated during the sonication process.We propose an equilibrium-based hypothesis to describe the coex-istence of the two phenomena (Fig. 7). Before sonication, DNAbinds SWNTs non-covalently as described in previous literature(b and c) [1]. Sonication of the solution causes a shift in equilib-rium by forming free radicals in solution creating covalent anchorsof the DNA to the SWNT (d). Multiple anchored features allow forthe dispersion in aqueous solution as well as non-covalent binding

interactions that are not as strong. Centrifugation (e and f) and fil-tration (g) steps necessary for purification of the conjugates, re-move insoluble material, but also disrupt non-covalentinteractions between the DNA and SWNTs. It has been noted byChen and Zhang that T30 is removed from T30:SWCNT complexesby hybridization to complementary A30 in solution, and that as aresult the CNTs precipitate from solution [49]. More recent studieshave shown that the kinetics of the removal of the DNA is complex[8]. Rapid initial displacement of DNA by small surfactant mole-cules was interpreted as loss of non-covalently DNA from imper-fectly associated nanotubes. Subsequent slow release wasproposed to occur from those nanotubes to which the DNA se-quences were perfectly wound in tandem, but still non-covalentlybound. The rate of DNA loss was temperature dependent with sig-nificant loss only occurring above 70 �C. Although the scenario pro-posed by Roxbury et al. seems perfectly plausible an alternativeexplanation would be that some of the DNA is covalently cross-linked to the carbon nanotubes. Upon heating the covalent bondsare broken and the DNA released. As pointed out earlier the ther-mal reversibility of covalent cross-links to carbon nanotubes iswell established.

We also found that the T30:SWCNT hybridizes to A30, but inour experiments most of the complex remains in solution. Sincethe amount of dispersion depends on multiple factors, includingcarbon nanotube length, diameter, chirality, relative surface areaof DNA coated vs. uncoated CNT, pH and buffer, it is not unex-pected that one might observe qualitative differences in dispersion.Chen and Zhang did not consider the possibility of covalent linkageand hence did not analyze their CNTs nor did they quantify theamount of T30 that was removed from the CNTs on treatment with

Fig. 7. Hypothesis of an equilibrium shift by sonication and the introduction of covalent anchors of the ODN to the SWNTs. Carbon nanotubes aggregate in aqueous solution(a). The presence of DNA leads to an interaction with the nanotube that is reversible and weak (b and c). Sonication generates ‘‘hot spots’’ or covalent links allowing a stronger,more efficient dispersion (d). Centrifugation (e and f) and filtration (g) steps lead to the removal of insoluble materials and non-attached DNA molecules. Slow loss of non-covalently DNA can lead to free DNA and partial CNT aggregation (h and I).

B.D. Dolash et al. / Chemical Physics 413 (2013) 11–19 17

A30. As illustrated in Fig. 7(g–i), we expect that there will alwaysbe some DNA that is associated by non-covalent interactions andcan be lost either by competition (binding to a complementary se-quence in solution) or simple dissociation.

In summary, we have run a series of experiments showing, inparticular, that free radical scavengers such as ascorbic acid andTrolox can effectively prevent the conjugation of DNA to SWCNTs.That suggests a significant role of free radicals in the associationbetween these two entities. We have also shown that the CNT-con-jugated DNA is able to effectively hybridize to complementary DNAsequences. These results are compatible with a mechanism inwhich the DNA is anchored to the carbon nanotubes by one ormore covalent linkages. Significant additional experimentation willultimately be required to establish the details of this hypothesis.But at the very least our results provide a working hypothesis thatwill serve as a useful stepping stone towards an understanding ofthe precise mechanism of DNA:SWCNT association. Such studiesare important given the intense interest in the development of nu-cleic acid-carbon nanotube systems for future electronic and bio-logical applications.

4. Methods

4.1. Materials

SWCNTs were purchased from Carbon Nanotechnologies, Inc.,(Houston, TX) and used as-received. Oligodeoxyribonucleotideswere ordered from Integrated DNA Technologies, Inc., (Coralville,IA) and used as received. All other materials were purchased fromSigma–Aldrich.

4.2. Dispersion of DNA:SWCNT

SWCNTs (4 mg) and DNA (4 mg) was combined in piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer (8 mL, 1 mM, pH = 7)and placed in a 15 mL polypropylene centrifuge tube. The mixturewas placed on ice and sonicated for 120 min (Sonics Model VC 130)at 80% amplitude using a 6 mm diameter probe tip. The solutionwas then separated into seven 1 mL aliquots and centrifuged at16,000g for 90 min. The supernatant (900 lL) was carefullyremoved from each aliquot and combined into three MilliporeAmicon� Ultra-4 centrifugal filter devices (molecular weightcut-off 100 kDa). The samples were desalted according to themanufacturer’s protocol using ultra-pure water as the desaltingsolvent. The concentrated, desalted samples were collected, com-bined and stored at room temperature.

The concentration of the solutions were determined by obtain-ing absorbance values of each solution using a Cary 3 Bio UV–Vis-ible spectrophotometer (k = 750 nm). The amount of DNA bound tothe SWNCTs was determined by collecting the filtrate from thedesalting procedure and characterizing the filtrate using UV–visi-ble spectrophotometry. A scan from 200–800 nm shows that thesolution is clean from detectable levels of SWCNTs and had detect-able levels of DNA (k � 260 nm). The concentration of DNA re-tained in the filtrate can be calculated from the A260 value andsubtracted from the starting amount to obtain the amount ofDNA bound to the SWCNTs in solution.

4.3. Free radical inhibitors

When studying the effect of free racial inhibitors on theDNA:SWCNT association, 4 mg of either (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) or ascorbic acid(vitamin C) was added to the mixture before sonication. The proce-dure was carried out as stated above.

4.4. Hybridization of complementary sequences to DNA:SWCNTs

DNA:SWCNTs were incubated with a complementary DNA at a1:1 m ratio of the amount of DNA bound to the SWCNTs (deter-mined as described above). Samples were mixed gently at 60 �Cfor 20 min and slowly cooled to room temperature. Free DNAwas filtered using a Millipore Amicon Ultra-4 centrifugal filter de-vice (MWCO 100 kDa). The fluorescein-conjugated hybrids wereobserved using a Varian Cary Eclipse Fluorescence spectrophotom-eter using an excitation wavelength of 495 nm and reading at theemission wavelength of 520 nm.

4.5. X-ray photoelectron spectroscopy (XPS)

XPS data were obtained by a Kratos Ultra DLD spectrometerusing monochromatic Al Ka radiation (hm = 1486.58 eV). The surveyand high-resolution spectra were collected at fixed analyzer passenergy of 160 and 20 eV, respectively. The spectra were collectedat 0� photoemission angle in the respect to the surface normal.The atomic concentrations of the chemical elements in the near-surface region were estimated after the subtraction of a Shirley typebackground, taking into account the corresponding Scofield atomicsensitivity factors and inelastic mean free pass (IMFP) of photoelec-trons as a standard procedure of CasaXPS software. The binding en-ergy (BE) values referred to the Fermi level were corrected using theC 1s peak set at 284.45 eV, which is characteristic position forgraphite and CNT. As-received SWCNTs and sonicated SWCNTswere mounted on the conducting double side-sticking tape.SWCNTs sonicated with Trolox were analyzed without removingfrom the Biomax-100 filter. In this case a commercial Kratos chargeneutralizer was used. A typical resolution measured as a Full Widthat Half Maximum (FWHM) of the C 1s deconvoluted peaks wasapproximately 0.75 eV. The XPS spectra were fitted by CasaXPSsoftware (version 2.3.14) after the Shirley background subtractionassuming line shape to be a Gaussian–Lorentzian function.

4.6. Raman spectroscopy

The spectra were collected at two excitation wavelength, 514.5and 647.1 nm. The Raman spectrometer T64000 from Jobin YvonHoriba was used with a triple monochromator, confocal micro-scope, and liquid nitrogen cooled CCD camera. The spectra werecollected in a backscattering mode using 20 � 0.40NA objectives.The excitation source is an Ar/Kr mix laser from Melles Griot withthe laser bandpass filters from Kaiser Optical Systems. The spectralresolution is about 1.5 cm�1.

Acknowledgments

The authors would like to take this opportunity to thank theWalther Cancer Institute for financial support. We would also liketo thank Nanotec Electronica for the WSxM software that was usedto acquire and analyze the SPM data.

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